The Stoichiometry between MICU1 and MCU Determines the Different Mitochondrial Ca2+ Uptake Phenotypes in Heart and Liver Wednesday, February 11, 2015 609a like ursodeoxycholic acid (UDCA) and tauroursodeoxycholic acid (TUDCA), are cytoprotective and inhibit cell death. The mechanisms associated with these distinct effects are not entirely clear. However, the effect of hydrophilic bile acids seems to be related with the blockage of a series of processes that converge on mitochondrial damage. Bax is a pro-apoptotic protein that belongs to the superfamily of the Bcl-2 proteins and is involved in mitochondrial pore formation. Submicellar concentrations of cytoprotective bile acids have been shown to modulate Bax concentration in mitochondria, suggesting that these molecules may interact directly with the protein. In this study, our objective was to evaluate the affinity of bile acids to recombinant Bax protein, making use of fluorescence spectroscopy (FRET and fluorescence anisotropy), as well as Fluorescence Correlation Spectroscopy (FCS). Our results show that the cytoprotective bile acids UDCA and TUDCA associate with recombinant Bax protein with high affinity, while the cytotoxic bile acid DCA only seems to be able to adsorb to the protein with much lower affinity. Notably, the bind- ing site for UDCA seems to be located in a hydrophobic pocket of the protein. This interaction could be responsible for the disruption of Bax translocation to the mitochondrial outer membrane in the presence of UDCA and/or TUDCA. Supported from FCT/Portugal (Projects PTDC/QUI-BIQ/119494/2010 and RECI/CTM-POL/0342/2012). T.S. and F.F. acknowledges FCT grants SFRH/BD/92398/2013 and SFRH/ BPD/64320/2009 3071-Pos Board B501 MAC Inhibitors Neutralize the Pro-Apoptotic Effects of Tbid Pablo M. Peixoto1, Oscar H. Teijido2, Laurent M. Dejean3, Evgeny Pavlov4, Bruno Antonsson5, Kathleen W. Kinnally4. 1Natural Sciences, Baruch College - CUNY, New York, NY, USA, 2National Institute of Child Health and Human Development, Baltimore, MD, USA, 3 Chemistry, California State University of Fresno, Fresno, CA, USA, 4 New York University College of Dentistry, New York, NY, USA, 5 Merck Serono, Geneva Research Center, Geneva, Switzerland. Since our initial characterization of the iMACs, different di-bromocarbazole derivatives with anti-apoptotic function have been developed and tested in several mouse models of brain injury and neurodegeneration [13-21]. Owing to the increased therapeutic potential of anti-apoptotic di-bromocarbazole de- rivatives, we sought to expand our knowledge of the mechanism of action of these small molecule inhibitors. We investigated the kinetics of MAC inhibi- tion in mitochondria from wild type, Bak, and Bax knockout cell lines using patch clamp electrophysiology, fluorescence microscopy, ELISA, and quantita- tive western blot analyses. Our results show that iMACs work through at least two mechanisms: 1) by blocking relocation of the cytoplasmic Bax protein to mitochondria and 2) by disassembling Bax oligomers in the outer membrane. A comparison of the inhibitory effects over channel conductance and cyto- chrome c release suggests that the iMACs interacted with both Bax and Bak with similar kinetics. Interestingly, wild type mitochondria were more suscep- tible to inhibition than the Bak or Bax knockouts. A quantitative western blot analysis showed that wild type mitochondria had lower steady state levels of Bak, which suggests an uneven stoichiometry of the MAC components. 3072-Pos Board B502 Tyrosine Phosphorylation of Mitochondrial Ca2D Uniporter Regulates Mitochondrial Ca2D Uptake Jin O-Uchi, Stephen Hurst, Jyotsna Mishra, Xiaole Xu, Bong Sook Jhun, Shey-Shing Sheu. Depariment of Medicine, Center for Translational Medicine, Jefferson Medical College, Thomas Jefferson University, Philadelphia, PA, USA. Mitochondrial Ca2þ has a critical role for balancing cell survival and death. Ca2þ influx into mitochondrial matrix is mediated primarily by the mitochondrial cal- cium uniporter (MCU). However, the signaling pathways that regulate MCU channel functions via post-translational modifications of MCU are completely unknown. Here we show that adrenergic signaling induces MCU tyrosine phos- phorylation and accelerates mitochondrial Ca 2þ uptake in cardiac cells. Adren- ergic signaling induces activation of proline-rich tyrosine kinase 2 (Pyk2) and translocation into the mitochondrial matrix; enhancing the interaction between Pyk2 and MCU, which subsequently accelerates mitochondrial Ca2þ uptake via Pyk2-dependent MCU tyrosine phosphorylation. MCU contains 15 tyrosine res- idues (5 in the N-terminus, 0 in the pore-forming region, 4 in transmembrane do- mains and 6 in the C-terminus), which are conserved across all eukaryotic species. Among them, only 3 of these tyrosine residues (Y157 at N-terminus, Y288, and Y316 at C-terminus in mouse MCU) remained as potential phosphor- ylation candidate sites for protein tyrosine kinases using phosphorylation predic- tion programs. We mutated these tyrosine residues to phenylalanine and generated non-phosphorylation mimetic MCU mutants (MCU-YFs). We confirmed that only two tyrosine sites were phosphorylated in response to adren- ergic stimulation in situ using cell lines stably expressing MCU-YFs. In addi- tion, overexpression of these MCU-YFs failed to increase mitochondrial Ca 2þ uptake in response to cytosolic Ca 2þ elevation by thapsigargin, whereas wild- type MCU transfection dramatically accelerates mitochondrial Ca2þ uptake compared to non-transfected cells. In summary, MCU contains Pyk2-specific phosphorylation site(s) and Pyk2-dependent tyrosine phosphorylation of MCU can modulate its channel functions and regulate mitochondrial Ca 2þ uptake. 3073-Pos Board B503 Cardioprotective Roles of Neuronal Ca 2D Sensor-1 during Stress Tomoe Y. Nakamura-Nishitani1, Shu Nakao1, Shigeo Wakabayashi2. 1Molecular Physiology, Natl.Cer.Cardiovasc.Ctr., Suita, Japan, 2Cardiac Physiology, Natl.Cer.Cardiovasc.CtrWakabayshi, Suita, Japan. Dysregulation of Ca2þ homeostasis in cardiomyocytes often results in heart fail- ure. Identifying molecular targets that regulate cardiomyocyte survival is of ther- apeutic importance. Neuronal Ca 2þ -sensor-1 (NCS-1) is an EF-hand Ca 2þ - binding protein, which is important for excitable cell functions. We previously found that NCS-1-deficient (Ncs1�/�) mice had excess neonatal mortality (Circ. Res.2011).The aim ofthe present study isto examinewhether NCS-1plays beneficial roles in cardiac survival during stress and the possible mechanisms un- derlying these effects. Neonatal mouse ventricular myocytes orwhole hearts from wild-type (WT) and Ncs1 �/� mice were subjected to stressors, and the resistance to stress was evaluated. Ncs1 �/� mouse hearts were more susceptible to stress induced by oxidative impairment and ischemia-reperfusion injury. Stress- inducedactivationofphosphatidylinositol3-kinase (PI3K)/Akt signaling, a major survival pathway, was substantially reduced in the Ncs1�/�group, and overex- pressionofNCS-1 ortheconstitutiveactive formofAkt increased the survivalrate of Ncs1 �/� myocytes. Cellular ATP levels, as well as mitochondrial respiration rates (both basal and maximal O2 consumption) were significantly depressed in Ncs1 �/� myocytes; especially with oxidative stress. Furthermore, intracellular Ca2þ handling was more easily dysregulated in stressed Ncs1�/� myocytes than WT myocytes. Since NCS-1 levels were increased by stress, the data suggest that NCS-1 is a survival-promoting factor, which is upregulated by stress stimuli. Interestingly, however,supra-physiological NCS-1 expression was toxic to cells. Taken together, our data suggest that moderate NCS-1 expression during stress promotes cardiomyocyte survival by maintaining proper Ca 2þ handling, which is required for activation of Akt survival pathways and mitochondrial function. 3074-Pos Board B504 Initiation of Electron Transport Activity and a Decrease of Oxidative Stress Occur Simultaneously during Embryonic Heart Development Gisela Beutner, George A. Porter, Jr. Pediatrics, University of Rochester Medical Center, Rochester, NY, USA. Mitochondria in early embryonic hearts are not thought to produce ATP, yet they do produce reactive oxygen species (ROS) that regulate myocyte differen- tiation. To assess changes in ATP and ROS generation in the developing heart, we measured mitochondrial oxygen consumption, the activity of the complexes (Cx) 1 and 2 of the electron transport chain (ETC), ETC supercomplex assem- bly, and ROS in embryonic mouse hearts. At embryonic day (E) 9.5, mitochon- drial ETC activity and oxidative phosphorylation (OXPHOS) are not coupled, even though the ETC complexes are present. We show that Cx-1 is able to accept electrons from the Krebs cycle, but enzyme assays that specifically mea- sure electron flow to ubiquinone or Cx-3 show no activity at this early embry- onic stage. At E11.5, mitochondria appear functionally more mature; ETC activity and OXPHOS are coupled and respond to ETC inhibitors. In addition, the assembly of highly efficient respiratory supercomplexes containing Cx �1, �3, and �4, ubiquinone, and cytochrome c begins at E11.5, the exact time when Cx-1 becomes functional activated. At E13.5, ETC activity and OXPHOS of embryonic heart mitochondria are indistinguishable from adult mitochon- dria. In contrast, generation of reactive oxygen species (ROS), as measured with Amplex Red, is high at E9.5 and drops significantly by E11.5, coinciding with activation of the ETC. In summary, our data suggest that between E9.5 and E11.5 dramatic changes occur in the mitochondria of the embryonic heart, which result in a decrease of ROS generation and an increase in OXPHOS due to the activation of Cx-1 and the formation of supercomplexes. 3075-Pos Board B505 The Stoichiometry between MICU1 and MCU Determines the Different Mitochondrial Ca 2D Uptake Phenotypes in Heart and Liver Melanie Paillard, György Csordás, Tünde Golenár, Cynthia Moffat, Erin Seifert, György Hajnóczky. MitoCare Center, Pathology, Thomas Jefferson University, Philadelphia, PA, USA. Mitochondrial Ca2þ uptake is central to oxidative metabolism and cell death signaling. The first clues to its molecular mechanism have emerged from the recent identification of the mitochondrial Ca 2þ uniporter’s pore forming protein (MCU) as well as its regulators. Among the regulators, MICU1 shows striking 610a Wednesday, February 11, 2015 co-expression and co-evolution with MCU. MICU1 has been demonstrated to be a Ca 2þ -sensing protein, which both sets a threshold for low Ca 2þ concentration while it assures cooperative activation during high Ca2þ rises. Mitochondrial Ca2þ uptake shows tissue specific differences and interestingly, mRNA level for MICUs and MCUs also displays tissue specificity. We set out to investigate if the stoichiometry between MICU1 and MCU could account for the previously described differences between heart and liver in mitochondrial Ca 2þ uptake. Immunoblotting showed higher expression for all MICU1, MICU2 and MCU in mouse liver versus heart mitochondria, and a 4.5 fold higher MICU1 to MCU ratio in liver. In fluorometric measurements of mitochondrial Ca2þ uptake, heart mitochondria displayed a decreased threshold and lesser cooperativity compared to liver mitochondria. Additionally, NAD(P)H elevation was detect- able after exposure to moderate [Ca 2þ ] elevations only in heart mitochondria. Overexpression of MICU1 in the heart using AAV9-MICU1 tail-vein injection significantly increased the MICU1 protein level without any changes of MICU2 or MCU. This increased the MICU1 to MCU ratio in the heart and led to increased thresholding and cooperativity, reproducing the liver-like mito- chondrialCa2þ uptakephenotype.ViceversaMICU1downregulationintheliver has been shown to lower the threshold and cooperativity of mitochondrial Ca 2þ uptake in hepatocytes. Thus, heart and liver mitochondria show different levels of Ca 2þ threshold and cooperative activation of Ca 2þ uptake, which seem to result from differential quantitative relationship between MICU1 and MCU. 3076-Pos Board B506 ER Calcium Release is Tuned by Mitochondrial Redox Nanodomains David M. Booth1, Balázs Enyedi2, Miklós Geiszt2, Péter Várnai2, György Hajnóczky1. 1MitoCare Center, Pathology, Thomas Jefferson University, Philadelphia, PA, USA, 2Department of Physiology, Semmelweis University, Budapest, Hungary. Spatially and temporally controlled increases of H2O2 emerge as an intracellular signal. We hypothesized that ROS and Ca 2þ interact locally, in the restricted vol- ume of the ER-mitochondrial interface. These physically tethered structures host enrichments of ion transport proteins such as the IP3 receptor, which support elevated nanodomains of Ca2þ during signalling events and are sensitive to H2O2. We used the genetically encoded H2O2 sensor HyPer incorporated into an inducible linker system to probe the redox environment at the ER- mitochondrial interface in HepG2 cells. We found a moderately elevated H2O2 nanodomain which developes into a H2O2 transient following IP3 receptor- mediated ER Ca2þ release and mitochondrial Ca2þ uptake. Pharmacological in- hibition showed that the transient was dependent upon ER Ca2þ, mitochondrial membrane potential and functional electron transport chain. HyPer measure- ments of the mitochondrial intermembrane space revealed significantly elevated H2O2 within this volume. Using electron microscopy we found that HepG2 mito- chondria possess a cohort of dilated cristae, which disappeared following IP3- linked Ca2þ release. Paxilline that inhibits mitochondrial BKCa channels blocked the cristae reshaping and also abolished the H2O2 transient at the inter- face. Furthermore, paxilline caused suppression of the IP3-linked calcium signal, whereas interface targeted killer red, a photoactivated H2O2 source, induced sensitization to the IP3-linked agonist. We conclude that the intermembrane/ cristae volume of mitochondria represents an oxidized pool fed by the electron transport chain. Ca 2þ -uptake stimulates expansion of the mitochondrial matrix via Kþ and concomitant water uptake, squeezing the oxidized volume of the cristae to the interface. Here, a transient H2O2 nanodomain sensitizes IP3 recep- tors to further stimulation. We demonstrate a physiological setting where Ca2þ release may autoregulate using mitochondrial H2O2 released from the cristae. 3077-Pos Board B507 Reactive Oxygen Species (ROS) Suppress Mitochondrial Motility Valentina Debattisti, Masao Saotome, Sudipto Das, Gyorgy Hajnoczky. MitoCare Center, Thomas Jefferson University, Philadelphia, PA, USA. Mitochondrial distribution and transport play pivotal roles for many cellular functions, including cell differentiation, cell division to ensure proper inheri- tance, apoptosis, ATP supply at the local sites of demand, Ca 2þ buffering for intracellular Ca2þ homeostasis. We previously showed that mitochondrial motility (mito-motility) is regulated by the cytosolic Ca2þ concentration ([Ca2þ]c), providing the basis for a homeostatic circuit in which the organelles decrease their movements along microtubules to locally buffer high [Ca 2þ ]c and contribute to ATP supply. Mito- chondria are also a major site for production and scavenging of ROS that serve as both a messenger and regulator of calcium signaling and are particularly rele- vant for the control of mitochondrial function. Here we tested the hypothesis that ROS target mito-motility to control mitochondrial function. H9c2 myoblast cells were transfected with a mitochondrial matrix targeted YFP and then loaded with fura2, to monitor the mito-motility simultaneously with [Ca 2þ ]c. H2O2 (100 mM) caused a decrease in mito-motility (6458 %) and an elevation in [Ca 2þ ]c (from 5558 to 9158 nM) at the same time. When the cells were incu- bated in a Ca 2þ -free medium and were pretreated with thapsigargin to prevent Ca2þ entry and intracellular Ca2þ mobilization, respectively, H2O2 continued to inhibit the mito-motility dose-dependently without any changes in [Ca2þ]c. These results indicate that H2O2 can cause suppression of mito-motility through a Ca 2þ independent mechanism we are currently analyzing. 3078-Pos Board B508 Miro1 is Dispensable for Calcium-Mediated Inhibition of Mitochondrial Movement David B. Weaver1, Agnieszka Lewandowska2, Tammy T. Nguyen2, Valentina Debattisti1, Janet M. Shaw2, Gyorgy Hajnoczky1. 1MitoCare Center, Dept of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, PA, USA, 2Biochemistry, University of Utah School of Medicine, Salt Lake City, UT, USA. Miro1 and 2 are (Rhot1 and 2) are two highly similar GTPases that are bound to the surface of mitochondria and possess EF-hand calcium binding motifs. Several groups have reported that Miro is involved in mitochondrial motility and inheritance, and particularly its calcium regulation, but the roles of the two isoforms have not been established. Genetic deletion of Miro1 in mouse is lethal at birth (Nguyen et al., 2014) and fibroblasts (MEFs) derived from Miro1 KO em- bryos show abnormal mitochondrial distribution, but the calcium-dependent in- hibition of motility is unaffected and the respiratory and calcium buffering capacities are normal. Neuron-specific knockout of Miro1 leads to progressive deficits of upper motor neuron function, however mitochondria in processes of cortical neurons from Miro1 KO and wild-type embryos showed comparable calcium-sensitive motility inhibition. While no significant increase in Miro2 pro- tein was observed in Miro1 KO MEFs, these data raise two possibilities: Miro1 and 2 are interchangeable with regard to calcium regulation of mitochondrial motility or Miro2 is the key player in this regard. To finally resolve this question, we are in the process of generating Miro2 KO and Miro1/2 KO cell lines. 3079-Pos Board B509 Mitochondrial Fusion Dynamics in the Heart Veronica Eisner1, Ryan Cupo1, Erhe Gao2, György Csordás1, Lan Cheng3, Jessica Ibetti2, J. Kurt Chuprun2, Walter J. Koch2, György Hajnóczky1. 1MitoCare Center, Pathology, Thomas Jefferson University, Philadelphia, PA, USA, 2Center for Translational Medicine, Temple University School of Medicine, Philadelphia, PA, USA, 3Center for Translational Medicine, Thomas Jefferson University, Philadelphia, PA, USA. Heart physiology depends on oxidative metabolism that likely requires dy- namic and permanent reorganization of mitochondria by fusion and fission. To directly evaluate mitochondrial fusion dynamics in cardiomyocytes (CM), mitochondrial matrix-targeted photoactivatable GFP and DsRed were intro- duced either in vitro or in vivo by adenovirus and were followed by confocal microscopy. Four conditions were analyzed: 24 to 48 h cultured neonatal and in vitro transduced adult CM, and CM from in vivo infected rat hearts. In the latter case, CM were isolated 7-10 days after infection and were imaged promptly or 24-48 h post harvesting. Neonatal CM mitochondria form a highly connected network, whereas both in vitro and in vivo transformed cultured CM displayed only some connectivity. Impressively, in vivo transduced adult CM that were imaged promptly after harvesting, unveiled a significantly higher con- nectivity among mitochondria than the 24-48h cultured adult CM. Furthermore, fusion events (f.e./75 square micrometers/min) were almost absent in cultured in vitro transduced CM, meanwhile in vivo transduced and cultured CM showed 0.450.2 f.e./min, whereas isolated, freshly-imaged CM displayed 1.45 0.1 f.e./min. Imaging in perfused whole heart ex vivo, showed consider- able mitochondrial continuity and fusion activity in ventricular CM. To study more directly the role of CM’s contractile activity in mitochondrial fusion, CM were incubated with Verapamil (10mM), that blocked spontaneous contrac- tion and partially suppressed the fusion activity of mitochondria. Also, mito- chondrial fusion activity appeared to be higher after spontaneous contraction or short term field stimulation in isolated freshly-imaged CM. Thus, mitochon- dria are dynamic in both neonatal and adult CM, but under culture conditions, adult CM lose mitochondrial fusion activity. This might be at least in part, because cardiomyocyte contractile activity is altered in culture and contractions likely provide some factors to support mitochondrial fusion activity. 3080-Pos Board B510 Mechanistic Characterization of the Thioredoxin System in the Removal of Hydrogen Peroxide Venkat R. Pannala, Ranjan K. Dash. Bioengineering and Biotechnology, Department of Physiology, Medical College of Wisconsin, Wauwatosa, WI, USA. The thioredoxin system plays a critical role in the defense against oxidative stress by removing harmful hydrogen peroxide (H2O2). Specifically, MAC Inhibitors Neutralize the Pro-Apoptotic Effects of Tbid Tyrosine Phosphorylation of Mitochondrial Ca2+ Uniporter Regulates Mitochondrial Ca2+ Uptake Cardioprotective Roles of Neuronal Ca2+ Sensor-1 during Stress Initiation of Electron Transport Activity and a Decrease of Oxidative Stress Occur Simultaneously during Embryonic Heart De ... The Stoichiometry between MICU1 and MCU Determines the Different Mitochondrial Ca2+ Uptake Phenotypes in Heart and Liver ER Calcium Release is Tuned by Mitochondrial Redox Nanodomains Reactive Oxygen Species (ROS) Suppress Mitochondrial Motility Miro1 is Dispensable for Calcium-Mediated Inhibition of Mitochondrial Movement Mitochondrial Fusion Dynamics in the Heart Mechanistic Characterization of the Thioredoxin System in the Removal of Hydrogen Peroxide